Method for the preparation of low molecular weight porcine lympho-reticular polypeptides and formulations thereof

ABSTRACT

A method for preparation of low molecular weight porcine lympho-reticular polypeptides. The method comprises the enzymatic hydrolysis of a source of protein, wherein the source of protein comprises a blend of porcine liver and porcine spleen, with a first enzyme having proteolytic activity and a second enzyme having amylase activity.

FIELD OF THE INVENTION

The present invention relates to a method for the preparation of lowmolecular weight polypeptides. More particularly, the present inventionis related to an enzymatic hydrolysis method to produce low molecularweight lympho-reticular polypeptides.

BACKGROUND OF THE INVENTION

Since ancient times, proteins of animal and vegetal origin have beenused for nutritional and developmental purposes. In fact, diets withnormal intake of proteins ensure better development and functioning ofphysiological functions and organic defenses. Broths with specificprotein content, e.g., liver, spleen, bone marrow, and/or blood, havebeen used for centuries, and as recently as the nineteenth and twentiethcenturies, in order to accelerate convalescence in patients affected bya wide variety of pathologies. At the beginning of the twentiethcentury, various collaborators in Argentina began to use extracts fromhealthy tissues that they called lysates, obtained from differenttissues of the human body, for the treatment of diseases that resultedin nutrient deficiencies.

The experience of more than a century using extracts from healthytissues has demonstrated the excellent acceptance of proteins of animalorigin, such as cattle, goats, and pigs, in human beings and therecognizable benefits towards the overall recovery of a patient.

After World War II, cancer treatment in Europe and North America wasstill in its infancy. Both, chemotherapy with nitrogen mustards andtreatments with radiotherapy were used. They were dangerous, unsafe andhad the potential to produce severe deleterious effects for doctors andpatients. At that time, certain protein compounds obtained from spleniccell fragments of pig origin were found to provide energy and nutritionto the patients to whom such compounds were administered. Thesesubstances, called CT compounds and later SP1 compounds, fostered asignificant decrease of tumor cellular glycolysis and effectivelyreversed a series of carcinoma and sarcoma type injuries.

The capacity of the CT and SP1 compounds to stimulate the production ofT-lymphocytes, peripheral mononuclear cells and the release of intrinsicgamma-interferon, has led to the classification of these compounds asimmune-stabilizers, modulators of biological response mechanisms (BRMs)or acute phase reactants (APRs), which positively activate production ofNatural Killer Cells (NKC).

Due to the above-mentioned properties, the CT and SP1 compounds havebeen shown to be beneficial in a wide variety of immune-compromisedpathologies, such as, HIV, herpes I and 2, herpes zoster, hepatitis B,rheumatoid arthritis, Sjögren syndrome, and dysplastic and aplasticanemia of different etiology, etc.

CT and SP1 compounds were also introduced for use in active treatment oftumoral injuries. Later, with the systematic use of chemotherapy andradiation treatments, CT and SP1 have been used as adjuvant therapy inoncological processes of various kinds. Experimental and clinical use inEurope and recently in Asia has demonstrated that the CT and SP1compounds elicit useful therapeutic effects and have provided asignificant improvement in the parameters of body weight, painsensation, improvement of the general state, quality of life andincreased survival in cancer patients undergoing traditional therapiessuch as chemotherapy and radiotherapy.

Processes for extracting CT and SP1 kind compounds are usually carriedout through enzymatic hydrolysis of proteins in acidic media. The finalcomposition and, therefore, the use of the hydrolysates depends mainlyon the protein source, the type of protease used, the hydrolysisconditions and the degree of hydrolysis reached in the reaction.

A single mammalian tissue is typically used as the raw material for thehydrolysis process. Chinese patent application CN1634987A, for example,discloses a method of preparing polypeptide extracts from the spleentissue of a mammal, other than a human. The process includes removingfat from the mammalian tissue, freeze-thaw crushing, homogenizing,adjusting the pH, precipitation, heating, centrifugation, filtration andultrafiltration in order to obtain spleen polypeptide extract.

Chinese patent CN102093440B discloses a method for obtaining pig brainprotein hydrolysate and monosialoganglioside (GM1) from fresh pig brainthrough fractional extraction.

Chinese patent application CN106520877A discloses a method for preparingpig cerebral protein antioxidative peptide. The method comprisespretreatment of pig brain to obtain degreased cerebral protein powder,ultrasonic treatment of pig cerebral protein powder, enzymatichydrolysis to extract crude pig brain polypeptide, ultrafiltration withthe molecular cut off of 3-5 kDa, and purification through a DEAE-52fiber resin layer.

U.S. Pat. No. 5,607,840 discloses a method for the preparation ofprotein hydrolysate from mammalian tissue having an endothelial ormucosal component, being essentially the only tissue present insubstrate suspension. Said mucosa tissue is obtained from at least oneof intestines, intestinal mucosa, intestinal skin, trachea tissue, lungtissue, and liver tissue and being derived from at least one of pigs,cattle and sheep.

Chinese patent CN103589770B discloses an industrial method for enzymatichydrolysis of proteins, starting from porcine whole blood proteins asthe raw materials, in order to obtain peptides and amino acids.

Other authors refer to protein sources different than mammalian tissuesas the raw material for the hydrolysis process. European patentapplication EP0325986A2 discloses a method for hydrolysis of food gradeproteins of vegetable origin, particularly soy protein isolates, throughcontact of an aqueous suspension of the protein with a combination ofprotease from fungus of the species Aspegillus oryzae and an enzymaticextract of porcine pancreas.

International patent application WO2015/031762A1 discloses differentmethods for recovery of lipids, sugars, and proteins from microbialbiomass of certain microalgae by enzymatic digestion. The methodcomprises the steps of treating microalgae with enzymes to producedigested biomass.

U.S. Pat. No. 4,361,586 discloses a process for enzymatic hydrolysis offish or fish by-products characterized by the utilization of vacuumconditions during the hydrolyzing phase of the process to selectivelyremove the odoriferous compounds.

Most authors cite the exclusive use of enzymes with proteolytic activityin order to hydrolyze the protein source. German patent applicationDE3929090A1 discloses a process to produce a defined protein hydrolysateby using an effective in the acid range proteolytic enzymes, such aspepsin or acid proteinase of Aspergillus.

US patent application US20170313987A1 discloses a method ofmanufacturing pancreas Islet of Langerhans (IOL) mimetics, were theobtained product is hydrolysable by trypsin, cellulase, dextranase,gelatinase, pepsin, pancreatin, papain, or bromelain.

European patent application EP0320717A2 discloses the use of anenzymatic system obtained by extracting germinated sorghum seeds, forthe hydrolysis of protein material from plant and animal sources.

European patent EP0566877B1 relates to a method of enzymatic hydrolysisof proteins and an apparatus for carrying out the method. The methoddescribed in that application uses proteolytic enzymes selected from thegroup consisting of trypsin, chymotrypsin, pancreatin, bacterialproteases, fungal proteases, and mixtures thereof.

In summary, the references found in the state of the art use a specificsource of protein due to the importance of having a specific source ofprotein in the final extract. In case of extracts obtained frommammalian tissues, the references use of a single mammalian organ as thesource of protein for the process.

SUMMARY OF THE INVENTION

A method for preparation of low molecular weight porcinelympho-reticular polypeptides. The method comprises the enzymatichydrolysis of a source of protein, wherein the source of proteincomprises a blend of porcine liver and porcine spleen, with a firstenzyme having proteolytic activity and a second enzyme having amylaseactivity.

The blend of porcine liver and spleen, is present at a ratio of about2:8 to 8:2 respectively, preferably 4:6 to 6:4. In some embodiments, theenzyme having proteolytic activity is selected from the group consistingof pancreatin, papain, pepsin, or mixtures thereof. The enzyme havingamylase activity is diastase in some embodiments. In one embodiment, thepapain is used at an amount ranging from 0.5 to 2.0 ounces per elevenpounds of the blend of porcine liver and spleen. In other embodiments,the pepsin is used at an amount ranging from 1.5 to 3.5 ounces pereleven pounds of porcine glands. The pepsin may be provided in the formof anhydrous pepsin.

In some embodiments, the pancreatin is used at an amount ranging from0.05 to 1.0 ounces per eleven pounds of fresh porcine glands. Thediastase is used at an amount ranging from 0.1 to 1.5 ounces per elevenpounds of fresh porcine glands.

In some embodiments of the method further comprises a cleansing step inan aqueous surfactant solution containing benzalkonium chloride. Theenzymatic hydrolysis has a pH range of between 4.5 to 5.5. The pH rangeis maintained through the use of a weak organic acid, such as citricacid. In some embodiments, the method includes a step of adding methyl4-hydroxybenzoate and Propyl 4-hydroxybenzoate to the enzymatichydrolysis. In further embodiments, the enzymatic hydrolysis is carriedout at a temperature of 35° C. and 55° C.

One embodiment discloses a low molecular weight porcine lympho-reticularpolypeptides formulation, comprising low molecular weight porcinelympho-reticular polypeptides made in accordance with the methoddescribed here, a vitamin, an antioxidant, and zinc. The antioxidant, insome embodiments is alpha lipoic acid. The antioxidant in otherembodiments is selected from resveratrol, glutathione, quercetin orastaxanthin, or combinations thereof. The zinc is in the form of zincgluconate dihydrate or zinc glycinate chelate. The vitamin is selectedfrom vitamin C, vitamin D3, vitamin B1, vitamin B2, vitamin B3, vitaminB5, vitamin B6, vitamin B12, biotin, and folic acid, or combinationsthereof.

Another embodiment consists of a low molecular weight porcinelympho-reticular polypeptides formulation, comprising low molecularweight porcine lympho-reticular polypeptides made in accordance with themethod described herein, a vitamin, bovine thymus extract, ascorbicacid, resveratrol, and zinc. The vitamin is selected from vitamin C,vitamin D3, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6,vitamin B12, biotin, and folic acid, or combinations thereof. The zincis in the form of zinc gluconate dihydrate or zinc glycinate chelate.

A low molecular weight porcine lympho-reticular polypeptidesformulation, comprising low molecular weight porcine lympho-reticularpolypeptides, an antioxidant, alpha glycerylphophorylcholine, and zinc.The antioxidant is alpha lipoic acid. The antioxidant is selected fromresveratrol, glutathione, quercetin or astaxanthin, or combinationsthereof. The zinc is in the form of zinc gluconate dihydrate or zincglycinate chelate.

A low molecular weight porcine lympho-reticular polypeptidesformulation, comprising low molecular weight porcine lympho-reticularpolypeptides of as described herein, a vitamin, ascorbic acid, and zinc.The zinc is in the form of zinc gluconate dihydrate or zinc glycinatechelate. The vitamin is selected from vitamin C, vitamin D3, vitamin B1,vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin, andfolic acid, or combinations thereof.

Another embodiment, comprises a low molecular weight porcinelympho-reticular polypeptides formulation, comprising low molecularweight porcine lympho-reticular polypeptides described herein,chondroitin, an antioxidant, and glucosamine. The antioxidant is alphalipoic acid. The antioxidant can also be selected from resveratrol,glutathione, quercetin or astaxanthin, or combinations thereof.

A low molecular weight porcine lympho-reticular polypeptidesformulation, comprising low molecular weight porcine lympho-reticularpolypeptides of described herein, an antioxidant, L-Carnitine, avitamin, and magnesium chloride. The antioxidant is alpha lipoic acid oris selected from resveratrol, glutathione, quercetin or astaxanthin, orcombinations thereof. The vitamin is selected from vitamin C, vitaminD3, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitaminB12, biotin and folic acid, or combinations thereof. In one embodiment,any of the formulations described above further comprises an amino acid,such as L-Lysine.

These and other objects, features and advantages of the presentinvention will become clearer when the drawings as well as the detaileddescription are taken into consideration.

BRIEF DESCRIPTION OF THE DRAWINGS

The method described herein can be better understood, without furtherlimitation, in connection with the accompanying drawings in which:

FIG. 1 is a diagrammatic representation of one illustrative embodimentof a method for the preparation of low molecular weight porcinelympho-reticular polypeptides.

FIG. 2 is a diagrammatic representation of another illustrativeembodiment of a method for the preparation of low molecular weightporcine lympho-reticular polypeptides.

FIG. 3 shows the results of the cytotoxicity effect of the low molecularweight porcine lympho-reticular polypeptides. Absorbance measurements atλ=490 nm determined no differences in cell viability between the cellstreated with the product at and below 5 mg/mL and the non-treated andLPS activated MDM, suggesting that the product is non-cytotoxic at theexperimental concentrations of 5.0 mg/mL and below.

FIG. 4 shows the results of the anti-inflammatory effect of the lowmolecular weight porcine lympho-reticular polypeptides. The productshows a significant anti-inflammatory effect for 1.25-5.0 mg/mL.Concentrations below 1.25 mg/mL show no anti-inflammatory effect.

DETAILED DESCRIPTION OF THE INVENTION

A method for the preparation of low molecular weight porcinelympho-reticular polypeptides is described herein.

FIG. 1 is a diagrammatic representation of one illustrative embodimentof a method 1000 for the preparation of low molecular weight porcinelympho-reticular polypeptides. The examples below are illustrative of amethod 1000 for the preparation of low molecular weight porcinelympho-reticular polypeptides in accordance with at least oneembodiment.

Turning to the illustrative embodiment of FIG. 1, the method 1000 forthe preparation of low molecular weight porcine lympho-reticularpolypeptides includes a trimming step 1100, which consists of trimmingporcine glands to remove any fat and/or non-glandular tissue which mayremain attached thereto. In at least one embodiment, porcine glandscomprise a mixture of fresh porcine livers and fresh porcine spleens,and in still another embodiment, the porcine glands comprise about 60%by weight of fresh porcine livers and about 40% by weight of freshporcine spleens. In some embodiments that ratio of liver to spleenranges between 8:2 and 2:8, preferably between 6:4 and 4:6. In yet onefurther embodiment, an amount of about 6.6 pounds of fresh porcinelivers and 4.4 pounds of fresh porcine spleens are utilized in themethod 1000 for the preparation of low molecular weight porcinelympho-reticular polypeptides.

Liver and spleen were chosen as the source of protein because they aresecondary organs of the immune system in mammalians, so they areexpected to contain a high amount of proteins with activity over theimmune system. On the other hand, porcine origin was chosen because ithas been found a high degree of similarity between the human genome andthe pig genome, which reduces the probability of allergic reactions, orother incompatibilities and adverse effects when the obtainedpolypeptides are administered as a dietary supplement for humans.

As described in more detail below, the glands are further sized 1200 toappropriate volume for further digestion of 1.5 to 27 cm³ for liver and0.625 to 1.0 cm³ for spleen. The blended glands are then pretreated 1300and the pretreated glands are further subjected to grinding 1400. Thegrinded glands are then blended 1500 to create a homogeneous blend. Thetreated homogeneous blend is then subject to digestion 1700. Thedigested blend is then dried 1800. The dried product is further grinded1900 and packaged 2000.

As shown in FIG. 2, the present method 1000, in at least one embodiment,further comprises a cleansing step 1120 in which the trimmed porcineglands are cleaned. Trimmed porcine glands are cleansed in a dilute,i.e., 0.01% to 4.00% preferably 1.00% to 2.00% by weight, aqueoussolution of a cationic surfactants, such as cetrimide, or otherquaternary ammonium compounds, including benzalkonium chloride,benzethonium chloride, and other similar surfactants for a time ofbetween 30 and 90 minutes, more preferably for about 60 minutes.Benzalkonium chloride is a surfactant which acts to burst the membraneof any germs which may be present in the trimmed porcine glands, therebyassuring germ free porcine glands for further processing.

In at least one further embodiment, an irradiating step 1140 is used inwhich the cleansed porcine glands are irradiated with ultravioletradiation during storage and/or prior to further processing to assuregerm free porcine glands for further processing.

The present method 1000 further comprises a sizing step 1200 for thetrimmed/cleansed porcine glands, i.e., cutting the trimmed/cleansedporcine glands into small sized porcine glands pieces having a volume of1.5 to 27 cm³ for liver and 0.625 to 1.0 cm³ for spleen. The glands canbe cut in any shape appropriate for further processing. The glands canbe cut into cubes, spheres, or any other type of shape.

The method 1000 further comprises a pretreating step 1300, wherein thesized porcine glands are pretreated with an enzyme which degradesproteins, i.e., an enzyme having proteolytic activity. In at least oneembodiment, pretreatment step 1300 comprises soaking the sized porcineglands in an aqueous solution of papain. Papain is an enzyme thatinitiates the digestion of the porcine glands. In one embodiment, papainis added in a predetermined amount ranging from 0.5 to 2.0 ounces pereleven pounds of fresh porcine glands, in the form of an aqueoussolution prepared by diluting about one ounce of papain in aboutseventeen fluid ounces of purified water. In at least one furtherembodiment, the sized porcine glands are soaked in an aqueous papainsolution for a period of between 30 to 90 minutes, preferably for about60 minutes.

The present method 1000 further comprises a grinding step 1400 in whichpretreated porcine glands are grinded. In at least one embodiment, astainless-steel industrial grinder is employed to grind the pretreatedporcine glands. In one embodiment, the grinding step 1400 includesgrinding the pretreated porcine glands combined with the aqueous papainpretreatment solution.

The method 1000 for the preparation of low molecular weight porcinelympho-reticular polypeptides further comprises a blending step 1500 inwhich the ground porcine glands are blended until a homogenous blend isobtained. As with the grinding step 1400, a stainless-steel industrialgrade blender can be utilized in accordance with at least one embodimentfor blending the ground porcine glands.

The present method 1000 further comprises a treating step 1600 in whichthe homogenous blend is treated with one or more digestive agents. Theblend can be digested with either diastase, pepsin, or pancreatin, orany combination of such digestive enzymes. In at least one embodiment,the digesting step 1600 comprises mixing the homogeneous blend with apredetermined amount of digestive agents such as diastase, pepsin and/orpancreatin. In one embodiment, diastase is added in a predeterminedamount ranging from 0.1 to 1.5 ounces per eleven pounds of fresh porcineglands; pepsin is added in a predetermined amount ranging from 1.5 to3.5 ounces per eleven pounds of porcine glands; and pancreatin is addedin a predetermined amount ranging from 0.05 to 1.0 ounces per elevenpounds of fresh porcine glands, in accordance with one embodiment of thepresent method 1000.

In yet one further embodiment, pepsin is provided in the form ofanhydrous pepsin.

In at least one embodiment, the method 1000 comprises diluting thepredetermined amounts of diastase (an amylase activity enzyme), pepsinand/or pancreatin (proteolytic enzymes) in an amount of purified water.The pH of the aqueous pepsin and pancreatin solution is adjusted 1620 toabout 4.5 to about 5.5 before it is added to homogenous blend. A dilutedsolution of a weak organic acid, for example, citric acid in a rangefrom 1.5 to 5.5 ounces per eleven pounds of fresh porcine glands, isutilized to adjust the pH of the aqueous diastase, pepsin and/orpancreatin solution.

The present method further comprises a step for preventing hydrolysis ofthe proteins and microbiological contamination. This step includes theaddition of a mixture composed by methyl 4-hydroxybenzoate and Propyl4-hydroxybenzoate to the previous blend comprising the porcine glandsused as the source of protein and the digestive agents used as theenzymatic system. methyl 4-hydroxybenzoate is used in an amount rangingfrom 0.4 to 1.0 ounces per eleven pounds of fresh porcine glands and thePropyl 4-hydroxybenzoate is used in an amount ranging from 0.1 to 0.5ounces per eleven pounds of fresh porcine glands.

The method 1000 for the preparation of low molecular weight porcinelympho-reticular polypeptides further comprises a second digesting step1700, wherein the treated blend is transferred to a reactor, ordigester, and allowing the digestive agents to digest the treated blendin a controlled digester environment.

In accordance with one embodiment, the digesting step 1700 is carriedout at a predetermined temperature range 1720. In one embodiment, thepredetermined temperature range is between 35° C. and 55° C., preferablybetween 40° C. and 50° C. In a preferred embodiment, a temperature rangeduring the digesting step 1700 is between 43° C. and 47° C., and in onefurther embodiment, the predetermined temperature range of between 44°C. and 46° C.

In further embodiments, the pH of the treated blend in the digester isadjusted 1740 to a predetermined pH range. In one embodiment, thepredetermined pH range of between 3.8 and 7.2, preferably between 4 and7. In one further embodiment, the pH of the treated blend in thedigester has a predetermined pH range of between 4.5 and 5.5. In stillone further embodiment, the pH of the treated blend in the digester hasa predetermined pH range of about 5.5 to about 6.0.

In one embodiment, digesting step 1700 further comprises agitation 1760of the treated blend in the digester. In one embodiment, the agitation1760 comprises an agitation period followed by a period of repose, andin one further embodiment, an agitation 1760 comprises a continuousrepetitive cycle of an agitation period followed by a period of repose.In at least one embodiment, agitation 1760 of the treated blend in thedigester comprises providing an agitation period of from 4 to 8 minutes,preferably about six minutes followed by a period of repose of from 2 to6 minutes, preferably about four minutes. In yet one further embodiment,agitation 1760 of the treated blend in the digester comprises providinga continuous repetitive cycle of from 4 to 8 minutes, preferably a sixminute agitation period, followed by a 2 to 6 minutes, preferably aboutfour minute period of repose over a 22 to 26 hour, preferably atwenty-four hour period.

In at least one embodiment, the present method 1000 for preparing lowmolecular weight porcine lympho-reticular polypeptides comprises testing1780 protein and microbiological profiles of samples of the treatedblend obtained intermittently from the digester while digesting thetreated blend in the digester. Such testing 1780 allows verification ofthe concentration of porcine lympho-reticular polypeptides present, aswell as to verify that the treated blend has not been contaminated bymicrobes.

The present method 1000 for the preparation of low molecular weightporcine lympho-reticular polypeptides further comprises drying 1800 adigested blend. In at least one embodiment, drying 1800 the digestedblend comprises spreading the digested blend in thin layers onto thedrying trays which are then placed into a drying oven. In oneembodiment, the drying oven comprises dry air injection to facilitatedrying of the digested blend. In at least one embodiment, drying traysare constructed of stainless steel, and in one further embodiment, thedrying trays comprise a plurality of drying apertures formed therethrough to facilitate passage of heated dry injection air through thedigested blend to facilitate drying of the same. A temperaturecontroller is employed in at least one embodiment to maintain thetemperature in the drying oven in a range of between 35° C. and 55° C.,preferably between 40° C. and 50° C. In at least one embodiment, dryingthe digested blend 1800 comprises maintaining the temperature in adrying oven at about 50° C. for about eight hours.

Once the digested blend has been dried, the present method 1000comprises grinding 1900 the dried product. In at least one embodiment,grinding 1400 the dried product comprises placing the dried product intoa stainless-steel industrial grinder and rendering a fine homogenouspowder. In at least one further embodiment, the present method 1000comprises sieving 1920 the ground product through a sieve. The sieve maybe constructed of stainless steel, and in at least one embodiment,comprises a sieve size in the range of between 20 and 40 (standard USmeasurement).

The present method 1000 for preparing low molecular weight porcinelympho-reticular polypeptides further comprises packaging 2000 a lowmolecular weight porcine lympho-reticular polypeptide product, whereinthe sieved product is placed in opaque plastic bags and stored in aninert atmosphere, such as nitrogen. In at least one embodiment, thepackaged product is maintained at a maximum storage temperature of about30° C.

One exemplary embodiment disclosed herein is a low molecular weightporcine lympho reticular polypeptides prepared in accordance with themethod 1000 described herein.

Another product comprises a dried blend of porcine glands, namely liverand spleen, that have been processed in accordance with the methoddescribed herein.

With the foregoing understanding of the present method 1000, thefollowing examples are illustrative of some non-limiting specificembodiments.

EXAMPLES Example 1: Preparation of Low Molecular Weight PorcineLympho-Reticular Polypeptides

Ingredients/Reagents.

Fresh porcine liver (certified free of antibiotics and hormones). Freshporcine spleen (certified free of antibiotics and hormones).Benzalkonium chloride solution (1-2% concentration). Purified water.Papain (diluted in the purified water). Pepsin (anhydrous). Pancreatin.Diastase. Citric acid. methyl 4-hydroxybenzoate. Propyl4-hydroxybenzoate.

Equipment. Stainless steel meat grinder. Stainless steel industrialblender. Stainless steel digester with temperature, pH, and agitationcontrols. Drying oven with temperature and vacuum controls. Stainlesssteel drying trays. Stainless steel sieves. Scale. Laboratory glassware.pH meter/controller.

Storage Bags and Containers

Preparation.

Approximately 6.6 pounds of fresh porcine livers and 4.4 pounds of freshporcine spleens, both certified to be free of antibiotics and hormones,are weighed out and trimmed to remove any fat or foreign matter. Thetrimmed porcine livers and spleens are cleansed for about one hour in abenzalkonium chloride solution having a concentration of about one totwo percent, by weight.

Benzalkonium chloride is a surfactant which causes the membranes of anygerms present in the porcine livers or spleens to burst, therebyassuring germ free raw materials for use in the present process. Thetrimmed porcine livers and spleens may be irradiated with ultravioletlight to further assure that the porcine glands are germ free.

The cleansed porcine livers and spleens are cut into small pieces andpretreated for about one hour in about seventeen ounces of purifiedwater having about one ounce of papain previously diluted therein. Thepapain solution initiates the digestion process and softens the porcinelivers and spleens to facilitate further processing.

The pretreated porcine glands are ground 1400 along with the papainsolution utilizing a stainless steel industrial grinder, and the groundporcine glands are then blended 1500 together, once again, along withthe papain solution, in a stainless steel industrial blender until ahomogenous blend is obtained.

A stainless steel digester is preheated to a temperature of about 40°C., and the homogenous blend of the pretreated porcine glands is addedthereto with an aqueous solution comprising about 0.5 ounces ofdiastase, about 2.1 ounces of anhydrous pepsin and about 0.1 ounces ofpancreatin mixed therein. An amount of diluted citric acid is added asneeded to adjust the pH of the aqueous pepsin and pancreatin solution toa range of about 5.5 to about 6.0. A temperature controller is programedto maintain the treated blend in the digester at a temperature of about45° C. throughout the digestion process, which is about twenty-fourhours.

A pH controller may be utilized, and diluted citric acid is added asneeded throughout the digestions process to adjust the pH of the treatedblend in the digester in a range of about 5.5 to 6.0.

0.5 ounces of methyl 4-hydroxybenzoate and 0.2 ounces of Propyl4-hydroxybenzoate are then added to the stainless-steel digestercontaining the blend comprising the porcine glands and the enzymaticsystem.

An agitation regimen is implemented in the stainless-steel digester andconsists of about six minutes of agitation followed by about fourminutes of repose. This agitation 1760 regimen is continuously repeatedover the entire digestion process which, once again, is abouttwenty-four hours.

Samples are obtained periodically throughout the digestion process toverify the protein profile of the treated blend, as well as to conductmicrobiological tests to assure that the treated blend has not beencontaminated during the digestion 1700 step. In the event a pHcontroller is not employed, the pH of the periodic samples is measured,and the pH of the treated blend is adjusted 1740 as may be needed by theaddition of diluted citric acid.

After about twenty-four hours in the digester, the digested 1700 blendis spread onto stainless steel drying trays which are placed into adrying oven for drying 1800. The drying oven uses forced dry air and thedrying trays have numerous apertures to permit the forced airflowtherethrough to shorten the overall drying time required to about eighthours. A temperature controller maintains the temperature in the dryingoven at about 50° C. The temperature controller is further programmed toassure that at no time does the over dryer temperature exceed 50° C., asprotein degradation may occur at temperatures in excess of 50° C.

The dried product is placed into the stainless-steel industrial grinderto form a fine homogenous powder, which is passed through astainless-steel sieve having sieve size in the range of 20 to 40(standard US measurement). The sieved product is placed in opaqueplastic bags and stored in an inert atmosphere, such as nitrogen, and ismaintained at a maximum storage temperature of about 30° C. In oneembodiment of the method, a further step includes collecting a productof the hydrolysis reaction as low molecular weight porcinelympho-reticular polypeptides.

Example 2: Evaluation of Cytotoxicity and Anti-Inflammatory Effects ofLow Molecular Weight Porcine Lympho-Reticular Polypeptides

The cytotoxicity and anti-Inflammatory effects of low molecular weightlympho-reticular polypeptides obtained in the Example 1 were evaluatedin human cell cultures.

A Human Acute Monocytic Leukemia cell line-THP-1 was used for thepurpose of this study. Cells were maintained in log phase growth in 75cm2 Falcon cell culture flaks containing complete medium (CM): GibcoRPMI 1640-L-Glutamine medium supplemented with 10% fetal bovine serumand 100U/mL Gibco Pen Strep. Cell cultures were incubated at 37° C.under 5% CO2 in a humidified atmosphere.

THP-1 cells were seeded in 96 well plates with 200 μL CM containing5.0×105 cells and treated with 50 ng/mL PMA for 48 hours to inducedifferentiation into macrophage-like cells MDM, then washed 4× with CMand allowed to rest for 5 to 7 days with media exchanged for fresh mediaevery two days. MDM were stimulated with 2 μg/mL LPS (E. coli serotype055: B5 Ready-Made LPS solution 1 mg/mL, 0.2 μm filtered).

To test for cytotoxicity, the supernatant of the product partiallydissolved in 5 mL of culture media were added to MDM (approx. 1×106cells/well with 200 μL of CM) at concentrations 10 ng-200 mg in 96 wellplates. After 24 hours of incubation, 20 μL of Cell titer 96 Aqueous OneSolution MTS 5 mg/mL was added and incubated for an additional 4 hours.Dual absorbance readings were taken in an Epoch microplate reader atλ=490 nm and 650 nm.

Cytokines in culture supernatants fluids treated at productconcentrations of 10 ng-200 mg were assayed using complete TNF-α captureantibody Novex ELISA kits according to manufacturers' instructions.Cytokine concentrations were determined within 30 min of the stopreaction. Dual absorbance readings were taken in an Epoch microplatereader at 450 nm and 650 nm respectively.

Statistical analysis was performed using a two-tailed t-test whereindicated. p<0.05 was considered significant. Experiments andmeasurements were performed in duplicate.

At concentrations of 10 mg/mL to 200 mg/mL, the product resultedcytotoxic to differentiated macrophage-like THP-1 cells, while atconcentrations of 5 mg/mL to 10 ng, the product proved non-cytotoxicity.FIG. 3.

On the other hand, at concentrations of 5 mg/mL and below, the productshowed downregulation of TNF-α at specific concentrations. The productdown-regulates the expression of TNF-α in cells activated with 2 ug ofE. coli serotype 055:B5 endotoxin (LPS) at concentrations of 1.25-5.0mg/mL. The downregulation is significant at p<0.05. These resultssuggest that the product acts as an ant inflammatory at concentrationsranging from 1.25-5.0 mg/mL. FIG. 4.

The product obtained through the method 1000 disclosed in the presentinvention does not compromise cell viability at concentrations of 5mg/mL or below, but at the same time do display potentialanti-inflammatory effects at concentrations of 1.25 mg/mL to 5 mg/mL,enables the possibility of using it as a therapeutically active agent atconcentrations between 1.25 mg/mL to 5 mg/mL.

Examples of Formulations of the Product for Consumption

The low molecular weight porcine lympho-reticular polypeptides productof the method described above is provided for consumption in variousforms. The various formulations described here, in some embodiments, arepackaged in such forms as are understood to a person of ordinary skillin the art. In some instances, the formulations are packaged in capsulesusing acid resistant membranes that manage release of the peptides andprotect them from digestion in the stomach prior to adsorption furtherin the digestive system. The capsules are normal or enteric. Entericcapsules are preferred in some embodiments, although any appropriatemedium of delivery can be utilized.

Immune System—Peptides with Alpha Lipoic Acid

In one exemplary formulation, low molecular weight porcinelympho-reticular polypeptides product of the method described herein isformulated with one or more vitamins. In one embodiment, the formulationcomprises low molecular weight Porcine-Lympho-Reticular Polypeptides,Vitamin C (as calcium ascorbate), vitamin D3 (cholecalciferol), anantioxidant (such as alpha lipoic acid), and zinc glycinate. Otherembodiments include additional vitamins such as vitamin C (in a suitableform, such as calcium ascorbate), vitamin D3 (Cholecalciferol), vitaminB1 (thiamin, in a suitable form such as thiamine mononitrate), vitaminB2 (riboflavin), vitamin B3 (niacin), vitamin B5 (in a suitable form,such as d-calcium panthenate), vitamin B6 (Pyridoxine HCL), vitamin B12(Cyanocabalamin) 1% DCP, folic acid DCP 10%, biotin, and alpha lipoicacid, or any other suitable antioxidant such as resveratrol,glutathione, quercetin, astaxanthin with phospholipids, which improveadsorption. Some embodiments also contain amino acids such as L-Lysine(in the form of L-lysine hydrochloride). Zinc Glycinate Chelate 20% Zn.Mycrocrystalline Cellulose. Talc USP. Magnesium Vegetable Stearate.Silicon Dioxide.

In one embodiment the formulation is included in a capsule with thefollowing concentrations:

Ingredient Description Mg/capsule Low Molecular weight PorcineLympho-Reticular 200 Polypeptides Vitamin C (as calcium ascorbate) 62.11Vitamin D3 (Cholecalciferol) 100,000 iu/g 2.00 Vitamin B1 - Thiamin (asthiamin mononitrate) 0.94 Vitamin B2 (Riboflavin) 95% Powder 0.89Vitamin B3 (Niacin) 10.20 Vitamin B5 (as d-calcium pantothenate) 5.86Vitamin B6 (Pyridoxine HCL) 1.26 Vitamin B12 (Cyanocobalamin) 1% DCP0.30 Folic Acid DCP 10% 2.00 Biotin Powder (1%) 15.00 Alpha Lipoic Acid120.00 L-Lysine (as L-lysine hydrochloride) 50.00 Zinc Glycinate Chelate20% Zn 37.50 Microcrystalline Cellulose 56.00 Talc USP 6.60 NaturalVanilla Flavor 41.00 Magnesium Vegetable Stearate 6.50 Silicon Dioxide6.00

The formulation may be administered in various serving sizes. In onepreferred embodiment the serving size is 3 capsules.

Immune System—Immuno competent Lympho Reticular Polypeptides added withThymus extract and Resveratrol as antioxidant.

In another embodiment, low molecular weight porcine lympho-reticularpolypeptides product of the method described herein is formulated withbovine thymus extract, ascorbic acid, vitamin D3, Resveratrol, and zincgluconate dihydrate (other appropriate forms of zinc). Other embodimentscomprise one or more vitamins as describes above. The formulation, insome embodiments, also includes bovine thymus extract, ascorbic acid (inthe form of ascorbic acid DC97%). A formulation may also includeresveratrol 50%. The amino acid used in some embodiments is L-LysineMono HCL. The composition may also include Capsule “0” blue. Oneexemplary formulation is provided below:

Ingredient Description Mg/capsule Low Molecular weight PorcineLympho-Reticular 200 Polypeptides Bovine Thymus Extract 100 AscorbicAcid DC 97% 50 Vitamin D3 (Cholecalciferol) 100,000 iu/g 2 Vitamin B1(Thiamine Mononitrate) USP 98% 0.75 Vitamin B2 (Riboflavin) 95% Powder0.85 Vitamin B3 (Niacin) 10 Vitamin B5 (Pantothenic Acid USP) 5 VitaminB6 (Pyridoxine HCL) 1 Vitamin B12 (Cyanocobalamin) 1% DCP 0.003 FolicAcid DCP 10% 0.2 Biotin Powder (1%) 0.15 Resveratrol 50% 100 L-LysineMono HCL 50 Zinc Gluconate Dihydrate or zinc glycinate 6 Orange Flavor13 Sodium Bicarbonate Grade 2 6 Microcrystalline Cellulose 4.64Magnesium Vegetable Stearate 5 Capsule “0” Blue 100

As described in any of these examples, Zinc Gluconate dihydrate can bereplaced, in some embodiments, with zinc glycinate.

The formulation may be administered in various serving sizes. In onepreferred embodiment the serving size is 3 capsules.

Multipurpose product with immune system adjuvant function for brain andcognitive support

In some embodiments, the low molecular weight porcine lympho-reticularpolypeptides are provided in a formulation to provide brain andcognitive support. Such formulations include the low molecular weightporcine lympho-reticular polypeptides, alpha glycerylphosphorylcholine(Alpha GPC), alpha lipoic acid, and zinc glycinate. The formulation mayalso include microcrystalline cellulose, aerosil, talc, magnesiumstearate, zinc glycinate, and a flavoring agent, such as natural vanillaflavor. One exemplary formulation includes:

Ingredient Description Mg/capsule Low Molecular weight PorcineLympho-Reticular 205 mg Polypeptides Alpha GPC (alphaglycerylphosphorylcholine) 125 mg Alpha lipoic acid 100 mgMicrocrystalline Cellulose 67 mg ??? Aerosil 20 mg Talc 30 mg Magnesiumstearate 10 mg Zinc Glycinate 37 mg Natural Vanilla Flavor 6 mg

The formulation may be administered in various serving sizes. In onepreferred embodiment the serving size is 3 capsules.

A further embodiment is used as a granulated immune support forchildren.

A further embodiment, one formulation comprises the low molecular weightporcine-lympho-reticular polypeptide product described herein, ascorbicacid, vitamin D3, and zinc. In some embodiments, the vitamins and otherelements disclosed above are included in the formulation. Thisformulation also includes, in some embodiments, calcium (in the form ofcarbonate, monobasic calcium phosphate, tribasic calcium phosphate,calcium pantothenate). The formulation also includes fructose,maltodextrin, other flavoring and coloring agents.

One example of this formulation includes the following concentrations:

mg/capsule Ingredient Description Low Molecular weight PorcineLympho-Reticular 205.0 Polypeptides Ascorbic Acid DC 97% 1000 Vitamin D3(Cholecalciferol) 100,000 iu/g 2.0 Vitamin B1 (Thiamine Mononitrate) USP98% 0.40 Vitamin B2 (Riboflavin) 95% Powder 0.45 Vitamin B3 (Niacin) 4.0Vitamin B5 (Pantothenic Acid USP) 5.0 Vitamin B6 (as pyridoxinehydrochloride) 12 Folate (Vitamin B9) (as folic acid) 100.0 mcg VitaminB12 (Cyanocobalamin) 1% DCP 20 mcg L-lysine Mono HCL 50 Calcium (ascalcium carbonate, monobasic 50 calcium phosphate, tribasic calciumphosphate, calcium pantothenate) Zinc (as Zinc Gluconate Dihydrate) 30Fructose 3515 Sodium bicarbonate 50 Maltodextrin 50 Strawberry flavor 50Beet extract (color) 100 Aerosil 25 mg Microcrystaline cellulose 50 mgOther Ingredients Malic Acid Tartaric Acid Citric Acid Ascorbic AcidAspartic Acid Trisodium Citrate Tocopherols

The serving size is per “satchel” as a powder. The formulationsdescribed in this application can be provided in powder form inside ofgels and capsules. The formulation can also be provided in any otheracceptable delivery method as understood by a person of ordinary skillin the art, such as satchels, packets, even in solution.

Another embodiment for immune system and cognitive support has thefollowing concentrations:

Ingredient Description mg/capsule Low Molecular weight PorcineLympho-Reticular 200 mg Polypeptides Vitamin C 100 mg Alpha Lipoic Acid100 mg Microcrystalline Cellulose 50 mg Aerosil 20 mg Talc USP 30 mgMagnesium stearate (Vegetal) 10 mg Zinc Glycinate 37.5 mg NaturalVanilla Flavor 6 mg

The weight per capsule is equal to 554.5=/−5%

Another embodiment for joint and immune system support comprises lowmolecular weight porcine lympho-reticular polypeptides, chondroitin,glucosamine, and alpha lipoic acid. One exemplary embodiment, has thefollowing formulation:

Ingredient Description mg/capsule Low Molecular weight PorcineLympho-Reticular 150 mg Polypeptides Chondroitin 100 mg glucosamine 100mg Alfa lipoic acid 50 mg Microcrystalline Cellulose 40 mg Talc USP 30mg Magnesium stearate (vegetal) 30 mg Aerosil 200 30 mg

In one embodiment, the low molecular weight lympho reticularpolypeptides can be used as coadyuvants in the treatment of type 2diabetes and immune system support. The formulation comprises lowmolecular weight porcine lympho-reticular polypeptide, alpha lipoicacid, L-carnitine, vitamin C and magnesium chloride. It may includeother components, as provided below:

Ingredient Description mg/capsule Low Molecular weight PorcineLympho-Reticular 200 mg Polypeptides Alpha lipoic acid 50 mg L-Carnitine50 mg Folic acid 20 mcg Vitamin B12 50 mcg Vitamin C 50 mgMicrocrystalline Cellulose 10 mg Aerosil 200 30 mg Talc USP 30 mgMagnesium stearate (vegetal) 20 mg Magnesium chloride 30 mg

In a further embodiment, the low molecular weight lympho reticularpolypeptides made in accordance with the method described here are usedin a granulated form for adults with joint pain as an adjuvant for paintreatment and immune system support.

The following is an example of such adjuvant:

weight/capsule Ingredient Description Low Molecular weight PorcineLympho-Reticular 300 mg Polypeptides Chondroitin 400 mg glucosamine 500mg Magnesium chloride 30 mg Vitamin D3 20 mcg Vitamin C 300 mg L-Lysine(hydrohloride) 100 mg Zing glycinate 30 mg Alpha lipoic acid 50 mgMicrocrystalline Cellulose 50 mg Aerosil 200 11 mg Talc USP 50 mgMagnesium stearate (vegetal) 50 mg Strawberry flavor 73 mg Fructose 3515mg Sodium bicarbonate 50 mg Maltodextrin 50 mg Strawberry flavor 50 mgBeet extract (color) 100 mg Other Ingredients Malic Acid Tartaric AcidCitric Acid Ascorbic Acid Aspartic Acid Trisodium Citrate Tocopherols

In this embodiment, the dosage is two sachets per day.

Since many modifications, variations and changes in detail can be madeto the described embodiment of the invention, it is intended that allmatters in the foregoing description and shown in the accompanyingdrawings be interpreted as illustrative and not in a limiting sense.Thus, the scope of the invention should be determined by the appendedclaims and their legal equivalents.

1. (canceled)
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 5. (canceled) 6.(canceled)
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 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. A low molecular weight porcine lympho-reticularpolypeptides formulation, comprising: a low molecular weight porcinelympho-reticular polypeptide, a vitamin, an antioxidant, and zinc. 18.The low molecular weight porcine lympho-reticular polypeptidesformulation of claim 17, wherein the antioxidant is alpha lipoic acid.19. The low molecular weight porcine lympho-reticular polypeptidesformulation of claim 17, wherein the antioxidant is selected fromresveratrol, glutathione, quercetin or astaxanthin, or combinationsthereof.
 20. The low molecular weight porcine lympho-reticularpolypeptides formulation of claim 17, wherein the zinc is zinc gluconatedihydrate or zinc glycinate chelate.
 21. The low molecular weightporcine lympho-reticular polypeptides formulation of claim 17, whereinthe vitamin is selected from vitamin C, vitamin D3, vitamin B1, vitaminB2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin, and folicacid, or combinations thereof.
 22. A low molecular weight porcinelympho-reticular polypeptides formulation, comprising: low molecularweight porcine lympho-reticular polypeptide, a vitamin, bovine thymusextract, ascorbic acid, resveratrol, and zinc.
 23. The low molecularweight porcine lympho-reticular polypeptides formulation of claim 22,wherein the vitamin is selected from vitamin C, vitamin D3, vitamin B1,vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin, andfolic acid, or combinations thereof.
 24. The low molecular weightporcine lympho-reticular polypeptides formulation of claim 22, whereinthe zinc is zinc gluconate dihydrate or zinc glycinate chelate.
 25. Alow molecular weight porcine lympho-reticular polypeptides formulation,comprising: a low molecular weight porcine lympho-reticular polypeptide,an antioxidant, alpha glycerylphophorylcholine, and zinc.
 26. The lowmolecular weight porcine lympho-reticular polypeptides formulation ofclaim 25, wherein the antioxidant is alpha lipoic acid.
 27. The lowmolecular weight porcine lympho-reticular polypeptides formulation ofclaim 25, wherein the antioxidant is selected from resveratrol,glutathione, quercetin or astaxanthin, or combinations thereof.
 28. Thelow molecular weight porcine lympho-reticular polypeptides formulationof claim 25, wherein the zinc is zinc gluconate dihydrate or zincglycinate chelate.
 29. A low molecular weight porcine lympho-reticularpolypeptides formulation, comprising: a low molecular weight porcinelympho-reticular polypeptide, a vitamin, ascorbic acid, and zinc. 30.The low molecular weight porcine lympho-reticular polypeptidesformulation of claim 29, wherein the zinc is zinc gluconate dihydrate orzinc glycinate chelate.
 31. The low molecular weight porcinelympho-reticular polypeptides formulation of claim 30, wherein thevitamin is selected from vitamin C, vitamin D3, vitamin B1, vitamin B2,vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin, and folic acid,or combinations thereof.
 32. A low molecular weight porcinelympho-reticular polypeptides formulation, comprising: a low molecularweight porcine lympho-reticular polypeptide, chondroitin, anantioxidant, and glucosamine.
 33. The low molecular weight porcinelympho-reticular polypeptides formulation of claim 32, wherein theantioxidant is alpha lipoic acid.
 34. The low molecular weight porcinelympho-reticular polypeptides formulation of claim 32, wherein theantioxidant is selected from resveratrol, glutathione, quercetin orastaxanthin, or combinations thereof.
 35. A low molecular weight porcinelympho-reticular polypeptides formulation, comprising: a low molecularweight porcine lympho-reticular polypeptide, an antioxidant,L-Carnitine, a vitamin, and magnesium chloride.
 36. The low molecularweight porcine lympho-reticular polypeptides formulation of claim 35,wherein the antioxidant is alpha lipoic acid.
 37. The low molecularweight porcine lympho-reticular polypeptides formulation of claim 35,wherein the antioxidant is selected from resveratrol, glutathione,quercetin or astaxanthin, or combinations thereof.
 38. The low molecularweight porcine lympho-reticular polypeptides formulation of claim 35,wherein the vitamin is selected from vitamin C, vitamin D3, vitamin B1,vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin andfolic acid, or combinations thereof.
 39. (canceled)
 40. The lowmolecular weight porcine lympho-reticular polypeptides formulation ofany of claim 32, wherein the amino acid is L-Lysine.